Coatings Containing Polymer Modified Enzyme For Stable Self-Cleaning Of Organic Stains

ABSTRACT

Temporary active coatings that are stabilized against inactivation by weathering are provided including a base associated with a chemically modified enzyme, and, optionally a first polyoxyethylene present in the base and independent of the enzyme. The coatings are optionally overlayered onto a substrate to form an active coating facilitating the removal of organic stains or organic material from food, insects, or the environment.

FIELD OF THE INVENTION

The present invention relates generally to coating compositionsincluding active substances and methods of their use to facilitateremoval of organic stains. In specific embodiments, the inventionrelates to compositions and methods for prevention of insect stainadherence to a surface as well as insect stain removal by incorporatinga chemically modified protein into base materials to degrade insect bodycomponents.

BACKGROUND OF THE INVENTION

Many outdoor surfaces are subject to stain or insult from naturalsources such as bird droppings, resins, and insect bodies. As a result,the resulting stain often leaves unpleasant marks on the surfacedeteriorating the appearance of the products.

Traditional self-cleaning coatings and surfaces are typically based onwater rolling or sheeting to carry away inorganic materials. These showsome level of effectiveness for removal of inorganic dirt, but are lesseffective for cleaning stains from biological sources, which consist ofvarious types of organic polymers, fats, oils, and proteins each ofwhich can deeply diffuse into the subsurface of coatings. Prior artapproaches aim to reduce the deposition of stains on a surface andfacilitate its removal by capitalizing on the “lotus-effect” wherehydrophobic, oleophobic and super-amphiphobic properties are conferredto the surface by polymeric coatings containing appropriatenanocomposites. An exemplary coating contains fluorine and siliconnanocomposites with good roll off properties and very high water and oilcontact angles. When used on rough surfaces like sandblasted glass,nanocoatings may act as a filler to provide stain resistance. A drawbackof these “passive” technologies is that they are not optimal for use inhigh gloss surfaces because the lotus-effect is based on surfaceroughness.

Photocatalytic coatings are promising for promoting self-cleaning oforganic stains. Upon the irradiation of sun light, a photocatalyst suchas Ti0₂ chemically breaks down organic dirt that is then washed away bythe water sheet formed on the super hydrophilic surface. As an example,the photocatalyst Ti0₂ was used to promote active fingerprintdecomposition of fingerprint stains in U.S. Pat. Appl. Publ.2009/104086. A major drawback to this technology is its limitation touse on inorganic surfaces due to the oxidative impairment of the polymercoating by Ti0₂. Also, this technology is less than optimal forautomotive coatings due to a compatibility issue: Ti0₂ not onlydecomposes dirt, but also oxidizes polymer resins in paint.

Therefore, there is a need for new materials or coatings that canactively promote the removal of organic stains on surfaces or incoatings and minimize the requirement for maintenance cleaning.

SUMMARY OF THE INVENTION

A process of facilitating the removal of organic stains is providedincluding providing a water-stabilized active temporary coating materialformed by associating a chemically modified enzyme with a base andcoating a substrate with the active coating material such that theenzyme is capable of enzymatically degrading a component of an organicstain in contact with the active coating material.

A water stabilized active temporary coating material is optionallycapable of degrading a component of an organic stain following immersionof said coating in water for 30 minutes or more, optionally where thecoating retains 50% or more activity following immersion in water for 30minutes.

A chemically modified enzyme is optionally a hydrolase such as abacterial neutral thermolysin-like-protease, an amylase, or a lipase.The enzyme is chemically modified by a polymeric moiety, optionally byat least one molecule of polyoxyethylene. The polyoxyethylene optionallyhas a molecular weight between 1,000 and 15,000 Daltons. In someembodiments, the polyoxyethylene further includes a succinimidyl esterprior to reaction with said enzyme. A polymeric moiety is optionallydirectly or indirectly covalently bound to an amino group on the enzymesuch as a terminal amino group or on a lysine. In some embodiments apolymeric moiety is directly or indirectly covalently bound to acysteine within the enzyme. It is appreciated that a polymeric moiety isoptionally linear or branched.

A water-stabilized active temporary coating material optionally iscovalently attached to at least one component of the base or isnon-covalently adhered to or admixed into the base. Such coatings whenpresent on a substrate optionally have a surface activity of 0.0075Units/cm² or greater when the coating includes a thermolysin as anenzyme.

The water-stabilized active temporary coating materials optionallyinclude a first polyoxyethylene associated with the base that isindependent of the enzyme. It is appreciated that the ratio of the baseto the enzyme in a coating is optionally 2:1 to 20:1 by weightrespectively. A composition optionally includes an enzyme that ischemically modified with a second polyoxyethylene. A first or secondpolyoxyethylene optionally has a molecular weight between 1,000 and15,000 Daltons. A first and second polyoxyethylene optionally have equalpolymers of oxyethylene. A first polyoxyethylene is optionallyderivatized sucha as with a succininimidyl ester. A secondpolyoxyethylene is optionally derivatized such as with a succininimidylester prior to reaction with an enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic for forming a water-stabilized active temporarycoating composition according to one embodiment of the invention;

FIG. 2 is a schematic of chemical modification of an enzyme and itsincorporation into a base according to one embodiment of the invention;

FIG. 3 illustrates homogenous incorporation of a chemically modifiedenzyme into a base according to one embodiment of the invention;

FIG. 4 illustrates water-stability of a coating incorporating achemically modified enzyme as measured by residual activity after waterwashing (A) or water contact angle before and after water washing (B);

FIG. 5 demonstrates facilitated removal of food stains on awater-stabilized active temporary coating after application to asubstrate.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following description of embodiment(s) of the invention is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly.

A composition useful as a coating is provided where enzymes associatedwith the coating material are modified so as to improve enzyme activitylifetime during and following exposure of a coating to water. Thecoatings provided herein are temporary coatings that have severaladvantages over other coating materials that are used to as a permanentcoating and are not intended to be renewed over the useful lifetime of acoated article. Temporary coatings are relatively simple to apply andcan be done by a layman in a home situation or by professionals. Use oftemporary coatings containing modified enzymes of the present inventionallows one to regularly renew the bioactive surface as well as improveother qualities such as shine, protection from the elements, and waterrunoff.

The coatings of the present invention demonstrate resistance to loss ofenzyme activity due to weathering. Weathering as defined herein includesexposure to water, heat, UV light, or other insult either in theenvironment or in a laboratory. Coatings according to the presentinvention have unexpected resistance to weathering by exposure to water,such as water immersion. As such, the term weathering includes immersionin water.

It is appreciated that the while the description herein is directed tocoatings, the materials described herein may also be substrates orarticles that do not require a coating thereon for promotion of organicstain removal. As such, the word “coating” as used herein means amaterial that is operable for layering on a surface of one or moresubstrates, or may comprise the substrate material itself. In someembodiments, a “coating” is exclusive of a substrate such that it is amaterial that may be used to overlay a substrate. As such, the methodsand compositions disclosed herein are generally referred to as an enzymeassociated with a coating for exemplary purposes only. One of ordinaryskill in the art appreciates that the description is equally applicableto substrates themselves.

The present invention is based on the catalytic activity of an enzyme toselectively degrade components of organic stains, thus, promoting activestain removal. Organic stains illustratively include organic polymers,fats, oils, or proteins. Inventive compositions and processes areprovided for the active breakdown of organic stains by awater-stabilized active temporary coating. Temporary coating materialsof the prior art have the capability to degrade organic stains, but theinventors unexpectedly discovered that, unlike permanent coatings, thesetemporary coatings are rapidly inactivated upon exposure to water suchthat the expected life of the coating is reduced to the point ofuselessness. Among the nearly infinite possible mechanisms of promotingenzyme stability, the inventors discovered that the addition of one ormore polymeric moieties on an enzyme prior to incorporation with a baseprovides for dramatically improved water-stability of the resultingcoating material.

As such, a water-stabilized active temporary coating materialcomposition is provided including a base with an associated chemicallymodified enzyme, and optionally a first polyoxyethylene also associatedwith the base, where the first polyoxyethylene is independent of theenzyme (i.e. not covalently linked to the enzyme). A composition hasutility as a coating for the self-cleaning of organic stains such asfood stains, insect stains, fingerprints, and other environmental orartificial insults.

A composition is a water-stabilized coating. The term “water-stabilized”denotes activity of the coating toward the self-cleaning or loosening ofan associated organic stain, where the activity is increased by thepresence of a chemically modified protein relative to the identicalcoating with a non-chemically modified protein. Water-stabilizedoptionally includes coatings that retain 50% to 90%, or any value orrange therebetween, or more activity after coating immersion in waterfor 30 minutes. Water-stabilized optionally includes coatings thatretain 15% or greater activity after coating immersion in water for 90minutes.

A composition is a temporary coating. As used herein the term“temporary” is defined as operable for a time between 30 minutes andthree months. It is appreciated that the outer limit of temporary isoptionally defined by the environmental conditions a coating issubjected to. Optionally, temporary is any time between application ofan inventive composition and subsequent immersion in or contact withwater. In some embodiments, temporary is at or less than three months,optionally, less than 2 months, optionally less than 6, 5, 4, 3, 2, or 1weeks, or any time or range of time therebetween. Optionally, temporaryis at or less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1day, or any time or range therebetween. In some embodiments, the term“temporary” is any time between application of an inventive compositionto a substrate and immersion or contact with water for 30, 60, or 90minutes, or more.

A composition includes a base material. As used herein a base is anycommercially or otherwise available automotive, furniture, floor, shoe,metal, or other surface conditioner, polish or protectant known in theart. Illustrative examples of a base include: naturally derived waxesillustratively paraffin wax, microcrystalline petroleum wax, carnaubawax, candelilla vegetable wax, montan coal derived wax; syntheticpolymeric waxes such as oxidized polyethylene; silicone-based waxesillustratively those found in U.S. Pat. No. 7,753,998,dimethylsilicones, aminofunctional silicones; a nonionic or anionicsurfactant in water composition illustratively that described in U.S.Pat. Nos. 5,073,407 and 5,968,238; and other materials commonly used forsurface conditioning, polish, or protection; and combinations thereof.

Specific examples of bases illustratively include polishes intended foruse on automobiles. Automobile polishes illustratively include: 1)TURTLE WAX carnauba car wax T-6 (carnauba wax containing silicone resinin petroleum distillates); 2) DURA SHINE as disclosed in U.S. Pat. No.5,073,407; 3) PLASTX a synthetic polymer auto polish; 4) TURTLE WAX ICEsynthetic polish which is a synthetic blend of hydrocarbons and siliconresins; 5) EAGLE ONE NANOWAX described in U.S. Pat. No. 7,503,963; 6) NUFINISH NF-76 as described in U.S. Pat. No. 7,067,573; and 7) TURTLE WAXPLATINUM series wax which is a blend of brazilian carnauba with bavarianmontan wax along with light reflective polymers, are purchased from alocal auto parts supplier.

A composition includes at least one active protein. An active protein isa macromolecule that has functional activity such as that of an enzymeillustratively a protease or hydrolase. A “protein” as defined herein asthree or more natural, synthetic, or derivative amino acids covalentlylinked by a peptide bond and possessing the activity of an enzyme.Accordingly, the term “protein” as used herein include between 3 andabout 1000 or more amino acids or having a molecular weight in the rangeof about 150-350,000 Daltons. A protein is a molecule with a contiguousmolecular sequence three amino acids or greater in length, optionallymatching the length of a biologically produced proteinaceous moleculeencoded by the genome of an organism. Examples of proteins include anenzyme, an antibody, a receptor, a transport protein, a structuralprotein, or a combination thereof. Proteins are capable of specificallyinteracting with another substance such as a ligand, drug, substrate,antigen, or hapten. It is appreciated that a protein is chemicallymodified by the addition of one or more homo or heteropolymeric moietiesas described herein. The term “analogue” is exclusive of chemicalmodification with a homo or heteropolymeric group with the exception ofbiotinylation.

A protein is optionally modified from a naked polypeptide sequence suchas by the addition or subtraction of one or more molecules ofphosphorus, sulfur, or by the addition of a pendent group such as abiotin, avidin, fluorophore, lumiphore, or other pendent group suitablefor purification, detection, or altering solubility or othercharacteristic of a protein.

The description herein is directed to a protein that is an enzyme, butit is appreciated that other protein active components are similarlyoperable herein. An enzyme is optionally a bioactive enzyme. A bioactiveenzyme is capable of cleaving a chemical bond in a molecule that isfound in a biological organism, the environment, or in food. An enzymeis optionally a protease that is capable of cleaving a peptide bondillustratively including a bacterial protease, or analogue thereof. Aprotein that functions as an enzyme is optionally identical to thewild-type amino acid sequence encoded by a gene, a functional equivalentof such a sequence, or a combination thereof. A protein is referred toas “wild-type” if it has an amino acid sequence that matches thesequence of a protein as found in an organism in nature. It isappreciated that a protein is optionally a functional equivalent to awild-type enzyme, which includes a sequence and/or a structural analogueof a wild-type protein's sequence and/or structure and functions as anenzyme. The functional equivalent enzyme may possess similar or the sameenzymatic properties as a wild-type enzyme, such as catalyzing chemicalreactions of the wild-type enzyme's EC classification, and/or maypossess other enzymatic properties, such as catalyzing the chemicalreactions of an enzyme related to the wild-type enzyme by sequenceand/or structure. An enzyme encompasses its functional equivalents thatcatalyze the reaction catalyzed by the wild-type form of the enzyme(e.g., the reaction used for EC Classification). As an illustrativenon-limiting example, the term “amylase” encompasses any functionalequivalent of an amylase that retains amylase activity though theactivity may be altered such as by increased reaction rates, decreasedreaction rates, altered substrate preference, increased or decreasedsubstrate binding affinity, etc. Examples of functional equivalentsinclude mutations to a wild-type enzyme sequence, such as a sequencetruncation, an amino acid substitution, an amino acid modification,and/or a fusion protein, etc., wherein the altered sequence functions asan enzyme.

An enzyme is either immobilized into or on coatings and catalyzes thedegradation of organic stain components into smaller molecules. Withoutbeing limited to one particular theory, the smaller product moleculesare less strongly adherent to a surface or coating such that gravity orgentle rinsing such as with water, air, or other fluid promotes removalof the organic stain material from the coating. Thus, the invention hasutility as a composition and method for the active removal of organicstains from surfaces.

Enzymes are generally described according to standardized nomenclatureas Enzyme Commission (EC) numbers. Examples of enzymes operable hereininclude: EC1, oxidoreductases; EC2, transferases; EC3, hydrolases; EC4,lyases; EC5, isomerases; or EC6, ligases. Enzymes in any of thesecategories can be included in a composition according to embodiments ofthe present invention.

In particular embodiments, an included enzyme is a hydrolase such as aglucosidase, a protease, or a lipase. Non-limiting examples ofparticular glucosidases include amylases, chitinase, and lysozyme.Non-limiting examples of particular proteases include trypsin,chymotrypsin, thermolysin, subtilisin, papain, elastase, andplasminogen. Non-limiting examples of lipases include pancreatic lipaseand lipoprotein lipase. Illustrative examples of proteins that functionas enzymes are included in U.S. Patent Application Publication No:2010/0210745.

Amylase is an enzyme present in some embodiments of a coatingcomposition. Amylases have activity that break down starch. Severaltypes of amylases are operable herein illustratively including α-amylase(EC 3.2.1.1) responsible for endohydrolysis of (1->4)-alpha-D-glucosidiclinkages in oligosaccharides and polysaccharides. α-Amylase isillustratively derived from Bacillus subtilis and has the sequence foundat Genbank Accession No: ACM91731 (SEQ ID NO: 1), or an analogue thereofand encoded by the nucleotide sequence of SEQ ID NO: 2. A specificexample is α-amylase from Bacillus subtilis available from Sigma-AldrichCo., St. Louis, Mo. Additional α-amylases include those derived fromGeobacillus stearothermophilus (Accession No: AAA22227), Aspergillusoryzae (Accession No: CAA31220), Homo sapiens (Accession No: BAA14130),Bacillus amyloliquefaciens (Accession No: ADE44086), Bacilluslicheniformis (Accession No: CAA01355), or other organism, or analoguesthereof. It is appreciated that β-amylases, γ-amylases, or analoguesthereof from a variety of organisms are similarly operable in aprotein-polymer composition.

Specific examples of amylase enzymes illustratively have 1000 U/gprotease activity or more wherein one (1) U (unit) is defined as theamount of enzyme that will liberate the non-protein digestion productform potato starch of Zulkowsky (e.g. starch, treated with glycerol at190° C.; Ber. Deutsch. Chem. Ges, 1880; 13:1395). Illustratively, theamylase has activity anywhere at or between 1,000 U/g to 500,000 U/g, orgreater. It is appreciated that lower activities are operable.

A protease is optionally a bacterial metalloprotease such as a member ofthe M4 family of bacterial thermolysin-like proteases of whichthermolysin is the prototype protease (EC 3.4.24.27) or analoguesthereof. A protease is optionally the bacterial neutralthermolysin-like-protease (TLP) derived from Bacillus stearothermophilus(Bacillus thermoproteolyticus Var. Rokko) (illustratively sold under thetrade name “THERMOASE C160” available from Amano Enzyme U.S.A., Co.(Elgin, Ill.)) or analogues thereof. A protease is optionally anyprotease presented in de Kreig, et al., J Biol Chem, 2000;275(40):31115-20. Illustrative examples of a protease include thethermolysin-like-proteases from Bacillis cereus (Accession No. P05806),Lactobacillis sp. (Accession No. Q48857), Bacillis megaterium (AccessionNo. Q00891), Bacillis sp. (Accession No. Q59223), Alicyclobacillisacidocaldarious (Accession No. Q43880), Bacillis caldolyticus (AccessionNO. P23384), Bacillis thermoproteolyticus (Accession No. P00800),Bacillus stearothermophilus (Accession No. P43133), Bacillus subtilis(Accession No. P06142), Bacillus amyloliquefaciens (Accession No.P06832), Lysteria monocytogenes (Accession No: P34025; P23224), amongothers known in the art.

A wild-type protease is a protease that has an amino acid sequenceidentical to that found in an organism in nature. An illustrativeexample of a wild-type protease is that found at GenBank Accession No.P06874 and SEQ ID NO: 3, with the nucleotide sequence encoding SEQ IDNO: 3 found in Takagi, M., et al., J Bacteriol., 1985; 163(3):824-831and SEQ ID NO: 4.

Methods of screening for protease activity are known and standard in theart. Illustratively, screening for protease activity in a proteaseprotein or analogue thereof illustratively includes contacting aprotease or analogue thereof with a natural or synthetic substrate of aprotease and measuring the enzymatic cleavage of the substrate.Illustrative substrates for this purpose include casein of which iscleaved by a protease to liberate folin-positive amino acids andpeptides (calculated as tyrosine) that are readily measured bytechniques known in the art. The synthetic substrate furylacryloylatedtripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtainedfrom Bachem AG, Bubendorf, Switzerland is similarly operable.

Specific examples of proteases illustratively have 10,000 Units/gprotease activity or more. In some embodiments, a protease is athermolysin wherein one (1) U (unit) is defined as the amount the enzymethat will liberate the non-proteinous digestion product from milk casein(final concentration 0.5%) to give Folin's color equivalent to 1 μmol oftyrosine per minute at the reaction initial reaction stage when areaction is performed at 37° C. and pH 7.2. Illustratively, the proteaseactivity is anywhere between 10,000 PU/g to 1,500,000 U/g inclusive orgreater. It is appreciated that lower protease activities are operable.Protease activity is optionally in excess of 300,000 U/g. Optionally,protease activity is between 300,000 U/g and 2,000,000 U/g or higher.

A protein is optionally a lipase. A wild-type lipase is a lipase thathas an amino acid sequence identical to that found in an organism innature. An illustrative example of a wild-type lipase is that found atGenBank Accession No. ACL68189 and SEQ ID NO: 5. An exemplary nucleotidesequence encoding a wild-type lipase is found at Accession No. FJ536288and SEQ ID NO: 6.

Lipase activity is illustratively defined in Units/gram. 1 Unitillustratively corresponds to the amount of enzyme that hydrolyzes 1μmol acetic acid per minute at pH 7.4 and 40 ° C. using the substratetriacetin (Sigma-Aldrich, St. Louis, Mo., Product No. 90240). The lipaseof SEQ ID NO: 5 may have an activity of 200 Units/gram.

Methods of screening for lipase activity are known and standard in theart. Illustratively, screening for lipase activity in a lipase proteinor analogue thereof illustratively includes contacting a lipase oranalogue thereof with a natural or synthetic substrate of a lipase andmeasuring the enzymatic cleavage of the substrate. Illustrativesubstrates for this purpose include tributyrin and triacetin both ofwhich are cleaved by a triacylglycerol lipase to liberate butyric acidor acetic acid, respectively, that is readily measured by techniquesknown in the art.

A protein optionally functions with one or more cofactor ions orproteins. A cofactor ion is illustratively a zinc, cobalt, or calcium.

Cloning, expressing, and purifying any protein operable herein isachievable by methods ordinarily practiced in the art illustratively bymethods disclosed in: Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992(with periodic updates); and Short Protocols in Molecular Biology, ed.Ausubel et al., 52 ed., Wiley-Interscience, New York, 2002.

Naturally derived amino acids present in a protein illustrativelyinclude the common amino acids alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, and tyrosine. It is appreciated that lesscommon derivatives of amino acids that are either found in nature orchemically altered are optionally present in a protein as well such asalpha-asparagine, 2-aminobutanoic acid or 2-aminobutyric acid,4-aminobutyric acid, 2-aminocapric acid (2-aminodecanoic acid),6-aminocaproic acid, alpha-glutamine, 2-aminoheptanoic acid,6-aminohexanoic acid, alpha-aminoisobutyric acid (2-aminoalanine),3-aminoisobutyric acid, beta-alanine, allo-hydroxylysine,allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylornithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxyisoquinoline, isovaline, 2-hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine.

A protein is obtained by any of various methods known in the artillustratively including isolation from a cell or organism, chemicalsynthesis, expression of a nucleic acid sequence, and partial hydrolysisof proteins. Chemical methods of protein synthesis are known in the artand include solid phase peptide synthesis and solution phase peptidesynthesis or by the method of Hackeng, T M, et al., Proc Nati Acad SciUSA, 1997; 94(15):7845-50. A protein may be a naturally occurring ornon-naturally occurring protein. The term “naturally occurring” refersto a protein endogenous to a cell, tissue or organism and includesallelic variations. A non-naturally occurring protein is synthetic orproduced apart from its naturally associated organism or is modified andis not found in an unmodified cell, tissue or organism.

Modifications and changes can be made in the structure of a protein andstill obtain a molecule having similar characteristics as an activeenzyme (e.g., a conservative amino acid substitution). For example,certain amino acids can be substituted for other amino acids in asequence without appreciable loss of activity or optionally to reduce orincrease the activity of an unmodified protein. Because it is theinteractive capacity and nature of a protein that defines that protein'sfunctional activity, certain amino acid sequence substitutions can bemade in a protein sequence and nevertheless obtain a protein with likeor other desired properties. Proteins with an amino acid sequence thatis not 100% identical to that found in nature are termed analogues. Ananalogue optionally includes one or more amino acid substitutions,modifications, deletions, additions, or other change recognized in theart with the proviso that any such change produces a protein with thesame type of activity (e.g. hydrolase) as the wild-type sequence. Inmaking such changes, the hydropathic index, or the hydrophilicity ofamino acids can be considered. In such changes, the substitution usingamino acids whose hydropathic indices or hydrophilicity values arewithin±2, those within±1, and those within±0.5 are optionally used.

Amino acid substitutions are optionally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include(original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),(Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly:Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg),(Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe),and (Val: Ile, Leu). In particular, embodiments of the proteins caninclude analogues having about 50%, 60%, 70%, 80%, 90%, 95%, or 99%sequence identity to a wild-type protein.

It is further appreciated that the above characteristics are optionallytaken into account when producing a protein with reduced or increasedenzymatic activity. Illustratively, substitutions in a substrate bindingsite, exosite, cofactor binding site, catalytic site, or other site in aprotein may alter the activity of the enzyme toward a substrate. Inconsidering such substitutions the sequences of other known naturallyoccurring or non-naturally occurring like enzymes may be taken intoaccount. Illustratively, a corresponding mutation to that of Asp213 inthermolysin is operable such as that done by Miki, Y, et al., Journal ofMolecular Catalysis B: Enzymatic, 1996; 1:191-199. Optionally, asubstitution in thermolysin of L144 such as to serine alone or alongwith substitutions of G8C/N60C/S65P are operable to increase thecatalytic efficiency by 5-10 fold over the wild-type enzyme. Yasukawa,K, and Inouye, K, Biochimica et Biophysica Acta (BBA)—Proteins &Proteomics, 2007; 1774:1281-1288. The mutations in the bacterial neutralprotease from Bacillus stearothermophilus of N116D, Q119R, D150E, andQ225R as well as other mutations similarly increase catalytic activity.De Kreig, A, et al., J. Biol. Chem., 2002; 277:15432-15438. De Kreigalso teach several substitutions including multiple substitutions thateither increase or decrease the catalytic activity of the protein. Id.and De Kreig, Eur J Biochem, 2001; 268(18):4985-4991. Othersubstitutions at these or other sites optionally similarly affectenzymatic activity. It is within the level of skill in the art androutine practice to undertake site directed mutagenesis and screen forsubsequent protein activity such as by the methods of De Kreig, Eur JBiochem, 2001; 268(18):4985-4991.

A protein is optionally an analogue of a wild-type protein. An analogueof a protein has an amino acid sequence that when placed in similarconditions to a wild-type protein possess some level of the activity ofa wild-type enzyme toward the same substrate. An analogue optionally has500%, 250%, 200%, 150%, 110%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 25%, 10%, 5%, or any value orrange of values therebetween, the activity of a wild-type protein. Anymodification to a wild-type protein may be used to generate an analogue.Illustratively, amino acid substitutions, additions, deletions,cross-linking, removal or addition of disulfide bonds, or othermodification to the sequence or any member of the sequence may be usedto generate an analogue. An analogue is optionally a fusion protein thatincludes the sequences of two or more wild-type proteins, fragmentsthereof, or sequence analogues thereof.

Methods of screening for protein activity are known and standard in theart. Illustratively, screening for activity of an enzyme illustrativelyincludes contacting an enzyme with a natural or synthetic substrate ofan enzyme and measuring the enzymatic cleavage of the substrate.Illustrative substrates for this purpose include casein, which iscleaved by a protease to liberate folin-positive amino acids andpeptides (calculated as tyrosine) that are readily measured bytechniques known in the art. The synthetic substrate furylacryloylatedtripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtainedfrom Bachem AG, Bubendorf, Switzerland is similarly operable.Illustrative substrates of α-amylase include long chain carbohydratessuch as amylose or amylopectin that make up starch. Other methods ofscreening for α-amylase activity include the colorimetric assay ofFischer and Stein, Biochem. Prep., 1961, 8, 27-33. It is appreciatedthat one of ordinary skill in the art can readily envision methods ofscreening for enzyme activity with the enzyme present in or on a varietyof materials.

A protein is illustratively recombinant. Methods of cloning,synthesizing or otherwise obtaining nucleic acid sequences encoding aprotein are known and standard in the art. Similarly, methods of celltransfection and protein expression are similarly known in the art andare applicable herein. Exemplary cDNA encoding the protein sequence ofSEQ ID NO: 1 is the nucleotide sequence SEQ ID NO: 2. Exemplary cDNAencoding the protein sequence of SEQ ID NO: 3 is the nucleotide sequencefound at accession number M11446 and SEQ ID NO: 4. Exemplary cDNAencoding the protein sequence of SEQ ID NO: 5 is the nucleotide sequenceSEQ ID NO: 6

A protein may be coexpressed with associated tags, modifications, otherproteins such as in a fusion protein, or other modifications orcombinations recognized in the art. Illustrative tags include 6XHis,FLAG, biotin, ubiquitin, SUMO, or other tag known in the art. A tag isillustratively cleavable such as by linking to protein via a targetsequence that is cleavable by an enzyme known in the art illustrativelyincluding Factor Xa, thrombin, SUMOstar protein as obtainable fromLifesensors, Inc., Malvern, Pa., or trypsin. It is further appreciatedthat chemical cleavage is similarly operable with an appropriatecleavable linker.

Protein expression is illustratively accomplished followingtranscription of a protein nucleic acid sequence, translation of RNAtranscribed from the protein nucleic acid sequence or analogues thereof.An analog of a nucleic acid sequence is any sequence that whentranslated to protein will produce a wild-type protein or an analogue ofa wild-type protein. Protein expression is optionally performed in acell based system such as in E. coli, Hela cells, or Chinese hamsterovary cells. It is appreciated that cell-free expression systems aresimilarly operable.

It is recognized that numerous analogues of protein are operable andwithin the scope of the present invention including amino acidsubstitutions, alterations, modifications, or other amino acid changesthat increase, decrease, or not do alter the function of the proteinsequence. Several post-translational modifications are similarlyenvisioned as within the scope of the present invention illustrativelyincluding incorporation of a non-naturally occurring amino acid,phosphorylation, glycosylation, addition of pendent groups such asbiotin, avidin, fluorophores, lumiphores, radioactive groups, antigens,or other molecules.

A protein according to the invention is chemically modified by theaddition of one or more polymeric moieties. Polymeric moietiesoptionally have a molecular weight ranging from 200 to 100,000 Daltons.Polymeric moieties are optionally linear, branched, liable, orcombinations thereof. The polymeric moieties are optionally homomeric orheteromeric. Illustrative examples of polymeric moieties include one ormore molecules of carbohydrate or polyoxyethylene (otherwise known aspolyethylene glycol or “PEG”).

Illustrative examples of polymeric moieties include but are not limitedto: polyalkyl alcohols and glycols (including heteroalkyl with, forexample, oxygen) such as polyoxyethylenes and polyoxyethylenederivatives; dextrans including functionalized dextrans; styrenepolymers; polyethylene and derivatives; polyanions including, but notlimited to, polymers of heparin, polygalacturonic acid, mucin, nucleicacids and their analogs including those with modified ribosephosphatebackbones, polypeptides of glutamate, aspartate, or combinationsthereof, as well as carboxylic acid, phosphoric acid, and sulfonic acidderivatives of synthetic polymers; and polycations, including but notlimited to, synthetic polycations based on acrylamide and 2-acrylamido-2methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similarquarternized polypyridine, diethylaminoethyl polymers and dextranconjugates, polymyxin B sulfate, lipopolyamines, poly(allylamines) suchas the strong polycation poly(dimethyldiallylammonium chloride),polyethyleneimine, polybrene, spermine, spermidine and proteins such asprotamine, the histone polypeptides, polylysine, polyarginine andpolyornithine; and mixtures and derivatives thereof. Suitable additionalpolymers are outlined in Roberts, M. J. et al. (2002) “Chemistry forpeptide and protein PEGylation” Adv. Drug Deliv. Rev. 54, 459-476;Kinstler, O. et al. (2002) “Mono-N-terminal poly(ethyleneglycol)-protein conjugates” Adv. Drug Deliv. Rev. 54; U.S. ApplicationSer. No. 60/360,722; U.S. Pat. No. 5,795,569; U.S. Pat. No. 5,766,581;EP 01064951; U.S. Pat. No. 6,340,742; WO 00176640; WO 002017;EP0822199A2; WO 0249673A2; U.S. Pat. No. 4,002,531; U.S. Pat. No.5,183,550; U.S. Pat. No. 5,985,263; U.S. Pat. No. 5,990,237; U.S. Pat.No. 6,461,802; U.S. Pat. No. 6,495,659; U.S. Pat. No. 6,448,369; U.S.Pat. No. 6,437,025; U.S. Pat. No. 5,900,461; U.S. Pat. No. 6,413,507;U.S. Pat. No. 5,446,090; U.S. Pat. No. 5,672,662; U.S. Pat. No.6,214,966; U.S. Pat. No. 6,258,351; U.S. Pat. No. 5,932,462; U.S. Pat.No. 5,919,455; U.S. Pat. No. 6,113,906; U.S. Pat. No. 5,985,236; WO9428024A1; U.S. Pat. No. 6,340,742; U.S. Pat. No. 6,420,339; and WO0187925A2.

Polyoxyethylene includes the generic structure —(CH₂CH₂O)_(n)—, where nis an integer optionally from 2 to 2000. Optionally, n is an integerranging from 50 to 500, optionally from 100 to 250, optionally from 150to 250. Polyoxyethylene is optionally provided in a range of sizesattached to proteins using one or more of a variety of chemistries knownin the art. Polyoxyelthylenes are optionally covalently associated withprimary amines (e.g. lysine side chains or the protein N-terminus),thiols (cysteine residues), or histidines. Lysine occurs frequently onthe surface of proteins, so binding of polyoxyethylene at lysine sidechains produces a mix of reaction products. Since the pKa of theN-terminus is significantly different than the pKa of a typical lysineside chain, it is possible to specifically target the N-terminus formodification. Similarly, as most proteins contain very few free cysteineresidues, cysteines (naturally occurring or engineered) may be targetedfor site-specific interactions with polyoxyethylene.

Polyoxyethylene is optionally end capped where one end is end-cappedwith a relatively inactive group such as an alkoxy group, while theother end is a hydroxyl group that may be further modified by linkermoieties. When the term “PEG” is used to describe polyoxyethylene theterm “PEG” may be followed by a number (not being a subscript) thatindicates a PEG moiety with the approximate molecular weight equal thenumber. Hence, “PEG10000” is a PEG moiety having an approximatemolecular weight of 10,000 Daltons. The inventors have found that someembodiments including linear PEG10000 are superior to other PEGmolecules.

The term “PEGylation” as used herein denotes modification of a proteinby attachment of one or more PEG moieties via a linker at one or moreamino acids. The polyoxyethylene (PEG) moiety is illustratively attachedby nucleophilic substitution (acylation) on N-terminal α-amino groups oron lysine residue(s) on the gamma-positions, e.g., with PEG-succinimidylesters. Optionally, polyoxyethylene moieties are attached by reductivealkylation—also on amino groups present in the protein usingPEG-aldehyde reagents and a reducing agent, such as sodiumcyanoborohydride. Optionally, polyoxyethylene moieties are attached tothe side chain of an unpaired cysteine residue in a Michael additionreaction using for example PEG maleimide reagents. Polyoxyethylenemoieties bound to a linker are optionally available from JenKemTechnology USA, Allen, Tex. It is appreciated that any PEG moleculetaught in U.S. Application Publication No: 2009/0306337 is operableherein. U.S. Application Publication No: 2009/0306337 also teachesmethods of attaching PEG groups to a protein. PEG is optionally linkedto a protein via an intermediate ester, amide, urethane, or otherlinkage dependent on the choice of PEG substrate and position ofmodification on a protein.

In some embodiments, a protein is an analogue of a hydrolase with theinclusion of additional cysteines to provide site specific incorporationsites for polyoxyethylene. In some embodiments, lysine or histidineresidues are substituted with alternative amino acids that do notpossess a target primary amine so as to prevent binding of a molecule ofpolyoxyethylene at that site. The choice of amino acid residues such ascysteines, lysines, or histidines to remove depends on the desiredextent of modification. Optionally, simulation computer programs areused to predict whether modification with a polymer will interfere withthe function of the protein as described in U.S. Pat. No. 7,642,340.

Proteins used in the inventions herein are optionally mono-substitutedi.e. having only one polymeric moiety attached to a single amino acidresidue in the protein molecule or to a N-terminal amino acid residue.Alternatively, two, three, four, or more polymeric moieties are presenton a single protein. In embodiments where protein includes more than onepolymeric moiety, it optionally has the same moiety attached to eachassociated amino acid group or to the N-terminal amino acid residue.However, the individual polymer groups may also vary from each other insize and length and differ between locations on the protein.

Reversible binding of one or more polymeric moieties at one or moresites on a protein is optionally used to regulate the rate of proteinleeching from a coating composition upon immersion in or contact withwater or other fluid. In these embodiments, the polymer is covalentlyattached but is liable upon exposure to weathering such as for exampleheating, water washing, or simply over time. The liable bond isoptionally the bond between the protein and the polymer or within alinker present between a protein and a polymer.

An inventive process uses an inventive composition that includes one ormore active chemically modified proteins incorporated into a base toform a coating material. The protein is optionally non-covalentlyassociated and/or covalently attached to the base material or isotherwise associated therewith such as by bonding to the surface or byintermixing with the base material during manufacture such as to produceentrapped protein. In some embodiments, the protein is covalentlyattached to the base material either by direct covalent interactionbetween the protein and one or more components of the base material orby association via a linker.

There are several ways to associate protein with a base in a coating.One of which involves the application of covalent bonds. Specifically,free amine groups of the protein are optionally covalently bound to anactive group of the base. Such active groups include alcohol, thiol,aldehyde, carboxylic acid, anhydride, epoxy, ester, or any combinationthereof. This method of incorporating protein delivers uniqueadvantages. First, the covalent bonds tether the proteins permanently tothe base and thus place them as an integral part of the finalcomposition with much less, if any at all, leakage of the protein.Second, the covalent bonds provide extended enzyme lifetime. Over time,proteins typically lose activity because of the unfolding of theirpolypeptide chains. Chemical binding such as covalent bondingeffectively restricts such unfolding, and thus improves the proteinlife. The life of a protein is typically determined by comparing theamount of activity reduction of a protein that is free or beingphysically adsorbed with that of a protein covalently-immobilized over aperiod of time.

A protein is optionally associated with a base at a ratio of 1:1 to 1:20(enzyme:base) by weight. Optionally, a protein is associated with a baseat a ratio of 1:2 to 1:15, optionally 1:4 to 1:12 by weight.

Proteins are optionally uniformly dispersed throughout the substratenetwork to create a homogenous protein platform.

Chemical methods of protein attachment to materials will naturally varydepending on the functional groups present in the protein and in thematerial components. Many such methods exist. For example, methods ofattaching proteins (such as enzymes) to other substances are describedin O′Sullivan et al, Methods in Enzymology, 1981; 73:147-166 andErlanger, Methods in Enzymology, 1980; 70:85-104.

Proteins are optionally present in a coating that is layered upon asubstrate wherein the protein is optionally entrapped in the basematerial, admixed therewith, modified and integrated into the basematerial or layered upon a base material.

A water-stabilized coating composition optionally includes one or moreadditives, optionally for modifying the properties of the compositionmaterial. Illustrative examples of such additives include one or morelight stabilizers such as a UV absorber or radical scavengerillustratively including those described in U.S. patent application Ser.No. 13/024,794 or U.S. Pat. No. 5,559,163, a plasticizer, a wettingagent, a preservative, a surfactant, a lubricant, a pigment, a filler,and an additive to increase sag resistance.

An inventive process optionally includes overlayering (coating) asubstrate with a water-stabilized active temporary coating material suchthat the protein is capable of enzymatically degrading a component of aorganic stain in contact with the active coating material. A substrateis any surface capable of being coated with an inventive coating. Asubstrate is optionally flexible or rigid with flexibility relative tothat of a polyvinylchloride sheet with a thickness of 10 mm. A substratehas a first surface and a second surface wherein the first surface andthe second surface are opposed. A coating is optionally overlayered on asubstrate on a first surface, a second surface, both, or fullyencapsulates a substrate. The coating of a substrate with awater-stabilized active coating material provides a self-cleaningsurface that promotes the removal or loosening of an organic stain whenpresent on the coating.

The identity of a substrate is limited only by its ability to be coatedwith an inventive composition. Illustratively, a substrate is metal,wood, natural or synthetic polymers such as fiberglass or otherplastics, resins, paints, lacquers, stone, leather, other material, orcombinations thereof. A substrate is optionally an automotive body panelor portion thereof. A substrate is optionally a boat hull or portionthereof. A substrate is optionally a wood floor or a coated wood floor.A substrate optionally includes a subcoating such as wood coated with apolyurethane protectant, or a subcoating is a paint, varnish, or otherprotectant commonly found on substrate. A water-stabilized temporaryactive coating material optionally contacts the substrate by overlayingthe subcoating material.

Water-stabilized coatings according to embodiments of the presentinvention provide good adhesion to substrates, protection againstenvironmental insults, protection against corrosion, and further provideactive properties of the protein. Thus, in certain embodiments, coatingsof water-stabilized active temporary material provide enzyme activity ona substrate useful in numerous applications such as detection of ananalyte which is a substrate for the enzyme or a ligand for a receptor,antibody or lectin. In particular embodiments, coatings provideresistance against staining by enzyme digestion of one or morecomponents of stain producing material.

When a water-stabilized composition is contacted with biological, food,or environmental material to produce an organic stain, the enzyme orcombinations of enzymes contact the stain, or components thereof. Thecontacting allows the enzymatic activity of the protein to interact withand enzymatically alter components of the stain improving its removalfrom the substrate or coating.

Proteins are included in compositions according to embodiments of thepresent invention in amounts ranging from 0.1-50, 1-30, 1-20, 1-10, 2-8,3-6, or other weight percent of the total weight of the materialcomposition.

Enzyme containing coatings have a surface activity generally expressedin Units/cm². Coatings including a thermolysin such as THERMOASE C160(thermolysin from Bacillus stearothermophilus) optionally havefunctional surface activities prior to exposure to water of greater than0.0075 Units/cm². In some embodiments, thermolysin surface activity isbetween 0.0075 Units/cm² and 0.05 Units/cm² or any value or rangetherebetween. Optionally, thermolysin surface activity is between 0.0075Units/cm² and 0.1 Units/cm² or any value or range therebetween.Optionally, thermolysin surface activity is between 0.01 Units/cm² and0.05 Units/cm² or any value or range therebetween. In coatingscontaining α-amylase from Bacillis subtilis, typical surface activitiesprior to exposure to water are at or in excess of 0.01 Units/cm². Insome embodiments, α-amylase surface activity is between 0.01 Units/cm²and 1.5 Units/cm² or any value or range therebetween. Optionally,α-amylase surface activity is between 0.01 Units/cm² and 2.5 Units/cm²or any value or range therebetween. Optionally, α-amylase surfaceactivity is between 0.01 Units/cm² and 1.0 Units/cm² or any value orrange therebetween. In some embodiments, α-amylase surface activity isat or between 0.01 Units/cm² and 4.0 Units/cm². It is appreciated thathigher surface activities are achievable by increasing the enzymeconcentration, using enzyme with a higher specific activity such as ananalogue of a wild-type enzyme, or by otherwise stabilizing enzymeactivity during association with a base material.

It is appreciated that the inventive processes of facilitating stainremoval will function at any temperature whereby the protein is active.Optionally, the inventive process is performed at 4° C. Optionally, aninventive process is performed at 25° C. Optionally, an inventiveprocess is performed at ambient temperature. It is appreciated that theinventive process is optionally performed from 4° C. to 125° C., or anysingle temperature or range therein.

The presence of protein combined with the material of a substrate or acoating on a substrate, optionally, with water or other fluidic rinsingagent, breaks down stains for facilitated removal.

An inventive process includes providing a coating with an enzyme suchthat the enzyme is enzymatically active and capable to cleave orotherwise modify one or more components of an organic stain. Inparticular embodiments, an organic stain is based on organic matter suchas that derived from an insect optionally an insect body, a fingerprint,foodstuffs, or from the environment.

An organic stain as defined herein is a stain, mark, or residue leftbehind after an organism, food, or environmental agent contacts acoating. An organic stain is not limited to marks or residue left behindafter a coating is contacted by an insect body. Other sources of organicstains are illustratively: insect wings, legs, or other appendages; birddroppings; food or components of food; fingerprints or residue leftbehind after a coating is contacted by an organism; or other sources oforganic stains such as bacteria or molecules present in water or soil.

Methods of preparing water-stabilized temporary active coating materialsillustratively include association of aqueous solutions of protein andcommercially available base materials by mixing such as by propellermixing or hand mixing to a uniform or a non-uniform distribution ofchemically modified protein to produce water-stabilized temporarycoating materials.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.

EXAMPLE 1

Materials for production of water-stabilized active temporary coatingmaterial.

Materials: Freeze-dried crickets are purchased from PetSmart. Cricketbodies reportedly contain 58.3% protein. (D. Wang, et al., EntomologicSinica, 2004; 11:275-283.) α-Amylase KLEISTASE SD80 from Bacillussubtilis (EC 3.2.1.1), lipase (lipase A12 (E.C.3.1.1.3) from Aspergillusniger), Protease N, Protease A, Protin SD AY-10, B. sterothermophilusTLP (THERMOASE C160), and THERMOASE GL30 (low activity preparation of B.sterothermophilus TLP) are obtained from AMANO Enzyme Inc. (Nagoya,JAPAN). Bovine serum albumin (BSA) from bovine serum, starch frompotatoes, starch from wheat, maltose, sodium potassium tartrate,3,5-dinitrosalicylic acid, Na₂(PO₄), NaCl, K₂(PO₄), casein,trichloroacetic acid, Folin & Ciocalteu' s phenol reagent, Na₂(CO₃),sodium acetate, calcium acetate, tyrosine, p-nitrophenyl palmitate,ethanol, iodine, glucose, maltose, maltotriose, maltohexose, dextrin (10kDa and 40 kDa) are obtained from Sigma Chemical Co., St. Louis, Mo.,U.S.A. Aluminum panels and 8-path wet film applicator are purchased fromPaul N. Gardner Company, Inc. (Pompano Beach, Fla.). Commercial basepreparations: 1) TURTLE WAX carnauba car wax T-6 (carnauba waxcontaining silicone resin in petroleum distillates); 2) DURA SHINE asdisclosed in U.S. Pat. No. 5,073,407; 3) PLASTX, a synthetic polymerauto polish; 4) TURTLE WAX ICE synthetic polish which is a syntheticblend of hydrocarbons and silicon resins; 5) EAGLE ONE NANOWAX describedin U.S. Pat. No. 7,503,963; 6) NU FINISH NF-76 as described in U.S. Pat.No. 7,067,573; and 7) TURTLE WAX PLATINUM series wax which is a blend ofbrazilian carnauba with bavarian montan wax along with light reflectivepolymers, are purchased from a local auto parts supplier. An Osterblender (600 watts) and light mayonnaise are obtained from a localsupermarket. Freeze-dried crickets are obtained from Fluker Laboratories(Port Allen, La.). Polyethylene glycol (PEG) derivatives withsuccinimidyl ester of different molecular weights are obtained fromFishersci (Pittsburgh, Pa.).

EXAMPLE 2

Preparation of Enzymes.

Lipase, α-amylase, and thermolysin are each prepared by ultrafiltrationfrom raw powder. For α-amylase, a 150 mL solution from raw powder (6.75g) is prepared in DI water. For thermolysin, a 150 mL solution of 1.5 gB. sterothermophilus thermolysin-like-protease (TLP) is prepared in DIwater. For lipase, a 150 mL solution of 1.5 g lipase A12 is prepared inDI water. The insoluble large impurity in raw powder is removed byfiltration over a 200 nm PTFE filter. The obtained solution has aprotein concentration of 20 mg/mL (measured by the Bradford method) andis maintained on ice.

Ultrafiltration is performed using a 150 mL Amicon cell (cooled withice) with a pressure of 55 psi and an ultrafiltration membrane with acut-off of 30 kDa from Millipore (Billerica, Mass.). Ultrafiltration isrepeated 3 times by refilling the cell back to 150 mL of DI water aftereach run. The final remaining purified protein solution is quantified bythe Bradford method and diluted to the final working concentration usedfor chemical modification and production of coating materials. Coatingsare made using a solution of 50, 100, 200, or 300 mg/mL of purifiedenzyme following chemical modification.

EXAMPLE 3

PEGylation of enzyme. Purified enzyme (c′-amylase, thermolysin, orlipase) is mixed with PEG (monofunctional linear PEG10000, PEG12000,PEG20000, or combinations) derivatized with succinimidyl ester at a moleratio of 1:5 enzyme:PEG in 0.05 M sodium phosphate buffer pH 7.5, andsubjected to mild agitation by shaking at 200 rpm at room temperaturefor 60 minutes. Some preparations further involve isolation ofnon-reacted PEG by filtration with a filter with an appropriatemolecular weight cut-off for each PEG used in the PEGylation reactions.

EXAMPLE 4

Commercial wax formulations 1-7 of Example 1 and a concentratedPEGylated-enzyme (200 mg/ml prior to PEGylation used without subsequentremoval of unreacted PEG) are vigorously mixed by vortexing at 1000 rpmat a ratio of from 4:1 to 12:1 base:enzyme by weight (unmodified enzyme)to form water-stabilized temporary coating material preparations usingthe base compositions of Example 1. Exemplary preparations are those ofA through KKK as depicted in Table 1.

TABLE 1 Preparation Base Enzyme Ratio (B:E) A 1 α-amylase 4:1 B 1α-amylase 8:1 C 1 α-amylase 12:1  D 1 thermolysin 4:1 E 1 thermolysin8:1 F 1 thermolysin 12:1  G 1 lipase 4:1 H 1 lipase 8:1 I 1 lipase 12:1 J 2 α-amylase 4:1 K 2 α-amylase 8:1 L 2 α-amylase 12:1  M 2 thermolysin4:1 N 2 thermolysin 8:1 O 2 thermolysin 12:1  P 2 lipase 4:1 Q 2 lipase8:1 R 2 lipase 12:1  S 3 α-amylase 4:1 T 3 α-amylase 8:1 U 3 α-amylase12:1  V 3 thermolysin 4:1 W 3 thermolysin 8:1 X 3 thermolysin 12:1  Y 3lipase 4:1 Z 3 lipase 8:1 AA 3 lipase 12:1  BB 4 α-amylase 4:1 CC 4α-amylase 8:1 DD 4 α-amylase 12:1  EE 4 thermolysin 4:1 FF 4 thermolysin8:1 GG 4 thermolysin 12:1  HH 4 lipase 4:1 II 4 lipase 8:1 JJ 4 lipase12:1  KK 5 α-amylase 4:1 LL 5 α-amylase 8:1 MM 5 α-amylase 12:1  NN 5thermolysin 4:1 OO 5 thermolysin 8:1 PP 5 thermolysin 12:1  QQ 5 lipase4:1 RR 5 lipase 8:1 SS 5 lipase 12:1  TT 6 α-amylase 4:1 UU 6 α-amylase8:1 VV 6 α-amylase 12:1  WW 6 thermolysin 4:1 XX 6 thermolysin 8:1 YY 6thermolysin 12:1  ZZ 6 lipase 4:1 AAA 6 lipase 8:1 BBB 6 lipase 12:1 CCC 7 α-amylase 4:1 DDD 7 α-amylase 8:1 EEE 7 α-amylase 12:1  FFF 7thermolysin 4:1 GGG 7 thermolysin 8:1 HHH 7 thermolysin 12:1  III 7lipase 4:1 JJJ 7 lipase 8:1 KKK 7 lipase 12:1 

The preparations of Table 1 are applied to various substrates byspreading with a cloth and optionally buffed as per the manufacturer'sdirections for each base preparation. Base formulation with free(unmodified) enzyme and base with unreacted PEG (no enzyme) are used ascontrols at the same conditions. Each material is spread on varioussurfaces including glass plates, quartz plates, plastics, aluminumpanels. When indicated by manufacturer of base, the coating is incubatedfor a “drying” period of 3 minutes followed by surface polishing withpaper towel by hand.

Each coating contains a uniform distribution of enzyme. Test panelscoated with preparations A-KKK are subjected to fluorescence microscopyto determine the uniformity of enzyme distribution. Test panels arecoated with coatings containing unmodified enzyme as a control (e.g.α-amylase non-chemically modified) or PEGylated enzyme. The intrinsicfluorescence of enzyme is used to detect the enzyme distribution.Coatings are applied to glass microslides and covered with a glassmicroslip and are subjected to light of wavelength 488 nm. Fluorescenceis detected at 570 nm using exposures of 5.313 seconds with a gain of 2.FIG. 3 depicts the distribution of preparation A at a magnification of200× indicating even distribution of enzyme in both preparations.Similar results are observed using preparations B-KKK.

EXAMPLE 5 Weathering Durability of Coatings

Coated aluminum panels are cut to test size of 1.2 cm×1.9 cm andsubjected to weather by submersion in room temperature DI water for 0minutes, 30 minutes, or 90 minutes with agitation. The test panels areremoved and rinsed with flowing DI water for 20 seconds followed byassay for remaining enzyme activity.

Test panels coated with α-amylase containing coatings are assayed bydetermination of amydolytic activity by reacting test panels with theα-amylase substrate 1% w/v potato starch in 20 mM sodium phosphatebuffer with 6.7 mM sodium chloride (pH 6.9). The substrate solution (2mL) is added to one rectangular piece of the coated test panel (1.2cm×1.9 cm) and incubated for 3 min at 25 ° C. The equivalent amount ofreducing sugar produced is determined using a UV-VIS spectrometer (Cary300-Varian Inc., Walnut Creek, Calif., USA) at 540 nm. One unit ofα-amylase activity is defined as 1.0 mg of reducing sugar (calculatedfrom a standard curve previously calibrated against maltose) releasedfrom starch in 3 min at room temperature.

Coatings prepared with thermolysin are assayed for proteolytic surfaceactivity essentially following the method of Folin and Ciocalteau, J.Biol. Chem., 1927; 73: 627-50. Briefly, 1 mL of 2% (w/v) casein insodium phosphate (0.05 M; pH 7.5) buffer solution is used as substratetogether with 200 μl of sodium acetate, 5 mM calcium acetate (10 mM; pH7.5). The substrate solution is pre-incubated in a water bath for 3 minto reach 37° C. The reaction is started by adding one piece of sampleplate coated with B. sterothermophilus TLP based coating (1.2×1.9 cm)followed by shaking for 10 min at 200 rpm at which time the reaction isstopped by adding 1 ml of 110 mM trichloroacetic acid (TCA) solution.The mixture is incubated for 30 min at 37° C. prior to centrifugation.The equivalent of tyrosine in 400 μL of the TCA-soluble fraction isdetermined at 660 nm using 200 μL of 25% (v/v) Folin-Ciocalteau reagentand 1 mL 0.5 M sodium carbonate. One unit of activity is defined as theamount of enzyme hydrolyzing casein to produce absorbance equivalent to1.0 μmol of tyrosine per minute at 37° C. This result is converted toUnits/cm².

An exemplary residual activity depicting the water-stability of coatingpreparations is depicted in FIG. 4A using preparation A (Table 1)compared to unmodified enzyme (free enzyme). Preparation A withPEGylated-enzyme shows approximately 70% and 30% activity at 30 minutesand 90 minutes, respectively, relative to coatings not subjected towater washing. The coatings containing unmodified enzyme (free enzyme)demonstrate less than 10% and 1.5% remaining after 30 and 90 minuteswater washing, respectively, relative to no water washing. This level ofwater-stability is also observed using preparations B-KKK of Table 1.Overall, these results indicate that coatings containing chemicallymodified enzyme are stable against water washing compared to coatingscontaining unmodified enzyme.

Water durability of PEGylated enzyme containing coatings is alsoobserved by retention of water contact angle. Water contact angle isused as a measure of the hydrophobicity or hydrophilicity of a surfacesuch as a coating. A highly hydrophilic surface will hold a waterdroplet with a contact angle typically of 0 degrees to 30 degrees. Ahighly hydrophobic surface will show contact angles of 90 degrees ormore. Changes in water contact angle represent changes in the chemicalmakeup of a surface or a coating. Water contact angle is measured onflat coated glass plates using a goniometer using the sessile dropmethod. As depicted in FIG. 4B, the absence of modification of an enzymeresults in nearly complete leeching of enzyme from the coating producinga reduction in water contact angle. In contrast, the water contact angleis maintained to a level lower than that for enzyme free base materialcoatings following the same water submersion conditions with estimated46% enzyme-PEG retention. (FIG. 4B.)

EXAMPLE 6

Preparation of organic stains and application to coated substrate andself-cleaning activity of coating preparations. For preparation ofinsect matter, 60 g of Freeze-dried crickets are chopped into powder bya blender (Oster, 600 watt) for 10 min. The stain solution is preparedby vigorously mixing 2 g of cricket powder with 6 mL of DI water. Atemplate of uniform spacing is used to apply the stain on the coatingsurface. The cricket stains are dried at 40 ° C. for 5 min. Each testpanel is placed into a glass dish subjected to rinsing with 200 mL of DIwater under 300 rpm shaking at RT for various times. The time of thestain removal is recorded. In order to reduce random error, the time ofthe first and last drop removed are not included. The average rinsingtime of eight stain spots is averaged for stain removal time. Testpanels coated with PEGylated thermolysin containing coatings provideimproved stain removal by gentle rinsing as compared to panels coatedwith base material alone.

Amylase containing coatings of Table 1 are placed on the plasticsurfaces of standard compact disks or aluminum test panels as in Example4. A 0.3 g sample of light mayonnaise is placed on various sections ofthe test panels followed by air dry at ambient conditions for 2 minutesprior to standing upright. Light mayonnaise includes largemacromolecules such as fat and starch that contribute to its highviscosity and thus to the high frictional force on the coating surfacethat prevents gravity driven sliding of the mayonnaise when the testpanel is tilted vertically. Coatings containing modified α-amylasecatalyze the hydrolysis of the emulsifier resulting in tremendouslylowered viscosity as a consequence of a phase separation at thestain-coating interface, thus allowing the stain to slide down the testpanel when tilted vertically.

Similarly, aluminum test panels are coated with preparation A of Table1, preparation A with PEG and no enzyme, or a coating with no enzyme orPEG as a control. As depicted in FIG. 5, the test panels coated withenzyme free coating (I) or PEG-coating (II) show no movement of thelight mayonnaise following tilting to a vertical position. The coatingwith PEGylated enzyme, however, shows significant self-cleaningremaining. (FIG. 5.)

The coatings of Table 1 containing PEGylated lipase are used to testremoval of fingerprints from glass or transparent plastic surfaces. Theself-cleaning of fingerprints by PEGylated lipase containingpreparations is tested at enzyme dilutions of 1:6 (I) and 1:12 (II)(Example 4)) on glass substrates. Test panels are coated with eitherPEGylated lipase containing base materials or control materials (noenzyme) and incubated at room temperature 24 hours. The test panels arestained with human fingerprints or facial skin contact. The coated testpanels are then incubated in an oven at 120° C. for 1 to 6 hours. Forbetter visualization of the remaining finger prints, coatings are washedunder running DI water (50 mL/sec) for 1 minute and dried using air.Prior to heating each coating is subjected to the same level of stainingby fingerprints. Following baking, coatings without enzyme showsignificant residual staining while coatings containing PEGylated lipaseshow greatly reduced stain remaining with the level of residualfingerprint staining reduced with increased enzyme concentration.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified or synthesized by one of ordinaryskill in the art without undue experimentation. Methods of proteinproduction and purification are similarly within the level of skill inthe art.

REFERENCE LIST

Harris, J. M. and Kozlowski, A. (2001). Improvements in proteinPEGylation: pegylated interferons for treatment of hepatitis C. J.Control Release 72, 217-224.

Veronese, F. and Harris, J. M. Eds. (2002). Peptide and proteinPEGylation. Advanced Drug Delivery Review 54(4), 453-609.

Veronese, F. M. ; Pasut, G. (2005), PEGylation, successful approach todrug delivery, Drug Discovery Today 10 (21): 1451-1458

Veronese, F. M.; Harris, J. M. (2002), Introduction and overview ofpeptide and protein pegylation, Advanced Drug Delivery Reviews 54 (4):453-456

Damodaran V. B. ; Fee C. J. (2010), Protein PEGylation: An overview ofchemistry and process considerations, European Pharmaceutical Review 15(1) : 18-26

Harris, J. M.; Chess, R. B. (2003), Effect of pegylation onpharmaceuticals. Nature Reviews Drug Discovery 2, 214-221

Rodriguez-Martinez J. A., et. al. (2008) Stabilization of a-ChymotrypsinUpon PEGylation. Correlates With Reduced Structural Dynamics.Biotechnology and Bioengineering, 101, 1142-1149

Li, J.; Kao, W. J. (2003), Synthesis of Polyethylene Glycol (PEG)Derivatives and PEGylated-Peptide Biopolymer Conjugates.Biomacromolecules 4, 1055-1067

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual patent or publicationwas specifically and individually incorporated herein by reference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A process of facilitating the removal of an organic stain on asubstrate comprising: associating a chemically modified enzyme with abase to form a water-stabilized active temporary coating material;coating a substrate with said active coating material such that saidenzyme is capable of enzymatically degrading a component of an organicstain in contact with said active coating material.
 2. The process ofclaim 1 wherein said water-stabilized active coating material is capableof degrading a component of an organic stain following immersion of saidcoating in water for 30 minutes or more.
 3. The process of claim 1wherein said active coating material retains 50% or more activityfollowing immersion in water for 30 minutes.
 4. The process of claim 1wherein said enzyme is a hydrolase.
 5. The process of claim 4 whereinsaid hydrolase is a bacterial neutral thermolysin-like-protease, anamylase, or a lipase.
 6. The process of claim 1 wherein said enzyme ischemically modified by the addition by at least one molecule ofpolyoxyethylene.
 7. The process of claim 6 wherein said polyoxyethylenehas a molecular weight between 1,000 and 15,000 Daltons.
 8. The processof claim 6 wherein said polyoxyethylene is covalently attached to saidenzyme via an intermediate urethane linkage.
 9. The process of claim 6wherein said polyoxyethylene is directly or indirectly covalently boundto an amino group on said enzyme.
 10. The process of claim 6 whereinsaid polyoxyethylene group is directly or indirectly covalently bound toa cysteine within said enzyme.
 11. The process of claim 6 wherein saidpolyoxyethylene is a branched molecule.
 12. The process of claim 1wherein said enzyme is an amylase and the surface activity of saidcoating is 0.01 units/cm² or greater.
 13. The process of claim 1 whereinsaid enzyme is covalently attached to at least one component of saidbase.
 14. The process of claim 1 wherein said enzyme is non-covalentlyadhered to or admixed into said base.
 15. A water-stabilized activecoating composition comprising: a base; a chemically modified enzymeassociated with said base; and a first polyoxyethylene associated withsaid base, said first polyoxyethylene independent of said enzyme;wherein said base, said enzyme, and said first polyoxyethylene form awater-stabilized active coating composition.
 16. The composition ofclaim 15 wherein said base and said enzyme are present in a ratio of 2:1to 20:1 by weight respectively.
 17. The composition of claim 15 whereinsaid enzyme is a hydrolase.
 18. The composition of claim 17 wherein saidhydrolase is a bacterial neutral thermolysin-like-protease, an amylase,or a lipase.
 19. The composition of claim 15 wherein said enzyme ischemically modified by covalent bonding to at least one molecule of asecond polyoxyethylene.
 20. The composition of claim 15 or 19 whereinsaid first polyoxyethylene has a molecular weight between 1,000 and15,000 Daltons.
 21. The composition of claim 19 wherein said firstpolyoxyethylene and said second polyoxyethylene have equal polymers ofoxyethylene.
 22. The composition of claim 15 wherein said firstpolyoxyethylene is derivatized with a succininimidyl ester.
 23. Thecomposition of claim 19 wherein said second polyoxyethylene iscovalently attached to said enzyme via an intermediate urethane linkage.24. The composition of claim 19 wherein said second polyoxyethylene isdirectly or indirectly covalently bound to an amino group on saidenzyme.
 25. The composition of claim 19 wherein said secondpolyoxyethylene is directly or indirectly covalently bound to a cysteinewithin said enzyme.
 26. The composition of claim 15 or 19 wherein saidfirst polyoxyethylene is a branched molecule.
 27. A biologically activewater-stable temporary self-cleaning surface comprising: thewater-stabilized active coating composition claim 15; and a substrate,wherein said water-stabilized active coating composition is overlayeredupon a surface of said substrate.
 28. The surface of claim 27 whereinsaid enzyme is a thermolysin and the surface activity of saidwater-stabilized active coating composition is 0.0075 units/cm² orgreater.